Clathrin-coated vesicle formation at the plasma membrane underlies the process of clathrin-mediated endocytosis by which the cell internalizes receptors, transmembrane channels, transporters, hormones growth factors and nutrients. Mis-regulation of endocytosis can cause neoplastic transformation. Therefore, the mechanism and regulation of clathrin-mediated endocytosis are of critical importance for human health. The early events of endocytosis involve formation of clathrin-coated pits, pit invagination, constriction and budding of the free vesicle. One of the essential factors required for endocytosis is dynamin, an unusual GTPase whose mechanism of action remains uncertain. Recent evidence suggests that dynamin is a master regulator of endocytosis, using its GTPase cycle to recruit the protein machinery that executes the constriction of invaginated vesicles. This laboratory has now identified Hsc70 and its co-chaperone auxilin as two proteins that specifically interact with dynamin:GTP. Two domains within auxilin that interact with dynamin have been mapped, and immunodepletion of auxilin from an in vitro endocytosis assay inhibits formation of constricted vesicles. These results are consistent with Hsc70/auxilin being dynamin effector. Importantly, Hsc70/auxilin was already known to catalyze removal of the clathrin coat from the free vesicle. Therefore, auxilin and Hsc70 act at early (vesicle formation) and late (vesicle uncoating) stages of endocytosis. The results suggest a model in which many, or perhaps all, stages of enocytosis are driven by Hsc70/auxilin-mediated rearrangements of the clathrin coat. It is envisioned that the specific consequences of Hsc70/auxilin on the clathrin coat are regulated by interactions with dynamin. This proposal addresses the following questions: Specific Aim 1 uses a combination of electron microscopy and an auxilin-dependent in vitro endocytosis assay to identify all the steps (initiation, invagination, constriction, fission) in clathrin-coated vesicle formation that require auxilin. In Specific Aim 2, mutants of dynamin that are deficient in auxilin binding will be used to corroborate the model that auxilin is a dynamin effector. Specific Aim 3 examines the function of dynamin self-assembly by testing whether severe assembly mutants of dynamin support endocytosis. The assembly defects of the mutants will be confirmed in vivo using fluorescence lifetime imaging microscopy (FLIM). Specific Aim 4 builds on the finding that auxilin binds lipids and will test whether auxilin acts as a lipid phosphatase. The function of lipid binding/phosphatase activity by auxilin in endocytosis will be examined using a semi-permeable cell assay.